The Aerodynamics of Free-Flight Maneuvers in Drosophila

Using three-dimensional infrared high-speed video, we captured the wing and body kinematics of free-flying fruit flies as they performed rapid flight maneuvers. We then “replayed” the wing kinematics on a dynamically scaled robotic model to measure the aerodynamic forces produced by the wings. The results show that a fly generates rapid turns with surprisingly subtle modifications in wing motion, which nonetheless generate sufficient torque for the fly to rotate its body through each turn. The magnitude and time course of the torque and body motion during rapid turns indicate that inertia, not friction, dominates the flight dynamics of insects

The aerodynamics of hovering flight in Drosophila

Using 3D infrared high-speed video, we captured the continuous wing and body kinematics of free-flying fruit flies, Drosophila melanogaster, during hovering and slow forward flight. We then `replayed' the wing kinematics on a dynamically scaled robotic model to measure the aerodynamic forces produced by the wings. Hovering animals generate a U-shaped wing trajectory, in which large drag forces during a downward plunge at the start of each stroke create peak vertical forces. Quasi-steady mechanisms could account for nearly all of the mean measured force required to hover, although temporal discrepancies between instantaneous measured forces and model predictions indicate that unsteady mechanisms also play a significant role. We analyzed the requirements for hovering from an analysis of the time history of forces and moments in all six degrees of freedom. The wing kinematics necessary to generate sufficient lift are highly constrained by the requirement to balance thrust and pitch torque over the stroke cycle. We also compare the wing motion and aerodynamic forces of free and tethered flies. Tethering causes a strong distortion of the stroke pattern that results in a reduction of translational forces and a prominent nose-down pitch moment. The stereotyped distortion under tethered conditions is most likely due to a disruption of sensory feedback. Finally, we calculated flight power based directly on the measurements of wing motion and aerodynamic forces, which yielded a higher estimate of muscle power during free hovering flight than prior estimates based on time-averaged parameters. This discrepancy is mostly due to a two- to threefold underestimate of the mean profile drag coefficient in prior studies. We also compared our values with the predictions of the same time-averaged models using more accurate kinematic and aerodynamic input parameters based on our high-speed videography measurements. In this case, the time-averaged models tended to overestimate flight costs

Changes in Immune Cell Types with Age in Breast are Consistent with a Decline in Immune Surveillance and Increased Immunosuppression

A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based image analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary breast tissue preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm2) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2 macrophage density was higher than M1 macrophage density and this difference was due to higher density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age.publishedVersio

Discriminating External and Internal Causes for Heading Changes in Freely Flying Drosophila

As animals move through the world in search of resources, they change course in reaction to both external sensory cues and internally-generated programs. Elucidating the functional logic of complex search algorithms is challenging because the observable actions of the animal cannot be unambiguously assigned to externally- or internally-triggered events. We present a technique that addresses this challenge by assessing quantitatively the contribution of external stimuli and internal processes. We apply this technique to the analysis of rapid turns (“saccades”) of freely flying Drosophila melanogaster. We show that a single scalar feature computed from the visual stimulus experienced by the animal is sufficient to explain a majority (93%) of the turning decisions. We automatically estimate this scalar value from the observable trajectory, without any assumption regarding the sensory processing. A posteriori, we show that the estimated feature field is consistent with previous results measured in other experimental conditions. The remaining turning decisions, not explained by this feature of the visual input, may be attributed to a combination of deterministic processes based on unobservable internal states and purely stochastic behavior. We cannot distinguish these contributions using external observations alone, but we are able to provide a quantitative bound of their relative importance with respect to stimulus-triggered decisions. Our results suggest that comparatively few saccades in free-flying conditions are a result of an intrinsic spontaneous process, contrary to previous suggestions. We discuss how this technique could be generalized for use in other systems and employed as a tool for classifying effects into sensory, decision, and motor categories when used to analyze data from genetic behavioral screens

Age-related gene expression in luminal epithelial cells is driven by a microenvironment made from myoepithelial cells

Luminal epithelial cells in the breast gradually alter gene and protein expression with age, appearing to lose lineage-specificity by acquiring myoepithelial-like characteristics. We hypothesize that the luminal lineage is particularly sensitive to microenvironment changes, and age-related microenvironment changes cause altered luminal cell phenotypes. To evaluate the effects of different microenvironments on the fidelity of epigenetically regulated luminal and myoepithelial gene expression, we generated a set of lineage-specific probes for genes that are controlled through DNA methylation. Culturing primary luminal cells under conditions that favor myoepithelial propogation led to their reprogramming at the level of gene methylation, and to a more myoepithelial-like expression profile. Primary luminal cells' lineage-specific gene expression could be maintained when they were cultured as bilayers with primary myoepithelial cells. Isogenic stromal fibroblast co-cultures were unable to maintain the luminal phenotype. Mixed-age luminal-myoepithelial bilayers revealed that luminal cells adopt transcription and methylation patterns consistent with the chronological age of the myoepithelial cells. We provide evidence that the luminal epithelial phenotype is exquisitely sensitive to microenvironment conditions, and that states of aging are cell non-autonomously communicated through microenvironment cues over at least one cell diameter

Understanding Ductal Carcinoma In Situ behavior: Dissecting microenvironment-dependent changes in tumor-suppressive and tumor-promoting functions of myoepithelial cells in the breast

Central to our understanding of breast cancer – a disease of dysregulation, is first defining how the normal human breast functions in the context of its microenvironment. Breast architecture is a complex interaction of structure and function directed towards maintenance of a relatively stable equilibrium – a homeostasis, in the performance of specific physiological processes. Cells are not cell-autonomous, they interact not only with other cells but also their microenvironment – one that they continually shape through a complex feedback system of dynamic and reciprocal signaling. We argue that it is through this framework that we can start to address one of the central challenges in breast cancer research to understand the molecular mechanisms driving progression from benign to invasive.As molecular profiling of Ductal Carcinoma In Situ (DCIS) and Invasive Ductal Carcinoma (IDC) have shown no robust genetic differences between the cancers, it has been argued that invasiveness is not a property acquired by the tumor, but rather a functional consequence of the loss of tumor-suppressive properties of the underlying supporting cells, specifically the myoepithelial cells that lie basally to the tumor cell-of-origin. Myoepithelial cells have been shown to be key regulators of normal tissue architecture, and dysregulation in their function can lead to loss of apico-basal polarity of luminal cells and changes in the deposition of basement membrane components (Gudjonsson, T., et al. 2002). Furthermore, in a xenograft model of DCIS, normal myoepithelial cells have been shown to have tumor-­suppressive properties, able to delay both tumor growth and acquisition of an invasive phenotype (Hu, M., et al. 2008). On the other hand, a transcriptomic profiling study comparing different cell populations of the breast between normal, DCIS and IDC shows that the earliest genetic changes occurring between normal and DCIS tissue arise in the myoepithelial lineage – largely through the aberrant upregulation of genes for secreted factors including matrix-remodeling enzymes, tumor-promoting cytokines and signaling factors (Allinen, M., et al. 2004). Thus, myoepithelial cells undergo a switch from tumor-suppressive to tumor-promoting behavior from normal to the earliest stage of cancer. When do these changes in the myoepithelial lineage begin to occur and what are the driving forces behind these changes? As changes in normal mammary gland architecture and its cellular and molecular composition are already apparent in aged breast tissue, including loss of lineage specificity in the epithelial lineages (Garbe, J.C., et al. 2012) (Benz, C.C. 2008), we propose that the underlying changes in the normal function of myoepithelial cells start to occur as early as aging, and play a key role in the processes underlying age-related susceptibility to breast cancer. Furthermore, we hypothesize that changes in the microenvironment that accompany aging and tumor progression drive these changes in the myoepithelial lineage through the process of “dynamic reciprocity,” where dynamic and reciprocal signaling between the altered myoepithelial microenvironment and the myoepithelial cells lead to further disruption of proper function of the lineage. To address these questions, we have (1) undertaken comprehensive transcriptomic profiling studies of myoepithelial cells in the context of normal primary breast tissue, and of an established human mammary epithelial cell (HMEC) culture system that is able to maintain the transcriptional, biochemical, and phenotypic features of lineage and chronological age (Garbe, J.C., et al. 2012); and (2) designed relatively simple yet biologically-relevant culture systems that allow for functional studies of myoepithelial cells in “young” vs. “aged” systems, and in “normal-like” vs. “tumor-like” microenvironments to illustrate how aberrations in normal myoepithelial function can lead to the changes we observe in aging and cancer.Our work to develop analytical methodologies that aim to move gene-level and cell-level analysis into a systems-level approach have generated a complex profile of the myoepithelial lineage: (1) Our transcriptomic profiling of primary myoepithelial cells, along with the other major cell populations of the breast, has elucidated major characteristics of myoepithelial cells including upregulated lineage-specific genes and their associated pathways, as well as potential cell-cell interactions and co-regulatory networks between myoepithelial cells and other cell populations in the breast, and their contribution to the maintenance of the tissue microenvironment; (2) Our transcriptomic profiling of myoepithelial cells isolated from HMEC derived from a cohort of young pre-menopausal and older post-menopausal women have identified key pathways, regulatory networks and transcription factors dysregulated in the myoepithelial lineage during aging. While our work to design biologically-relevant cell culture systems has revealed key insights into the phenotypic changes observed in aging and cancer: (1) our “young” and “aged” co-culture system illustrates the ability of myoepithelial cells to confer an age-specific phenotype to luminal cells; and (2) our “normal-like” and “tumor-like” culture system demonstrates the ability of extracellular matrix (ECM) proteins to differentially regulate the myoepithelial secretome, with “tumor-like” ECM inducing myoepithelial secretion of angiogenic, cancer-associated factors, and matrix remodeling enzymes that mimic their aberrant secretory behavior in DCIS.One the standing challenges of breast cancer research is to understand the underlying molecular mechanisms driving progression of breast cancer from benign to invasive. While progress has been made to identify tumor suppressor genes and oncogenes, as well as genetic alterations that associate with certain cancer subtypes, the correlation of these genotypic-phenotypic events is not fully understood. This poses a problem not only in understanding the biology of cancer, but equally significant, in developing reliable prognostic markers of progression and recurrence, as well as designing treatment options for women. Our current understanding of progression implicates myoepithelial cells as a major driver regulating transition to invasiveness. Thus, our understanding of how normal myoepithelial cells function, and how they become dysregulated in biologically-relevant model systems of aged and tumor-like microenvironments can provide insight into the molecular mechanisms which underlie the loss of normal function of the myoepithelial lineage that drive cancer progression

Understanding Ductal Carcinoma In Situ behavior: Dissecting microenvironment-dependent changes in tumor-suppressive and tumor-promoting functions of myoepithelial cells in the breast

Central to our understanding of breast cancer – a disease of dysregulation, is first defining how the normal human breast functions in the context of its microenvironment. Breast architecture is a complex interaction of structure and function directed towards maintenance of a relatively stable equilibrium – a homeostasis, in the performance of specific physiological processes. Cells are not cell-autonomous, they interact not only with other cells but also their microenvironment – one that they continually shape through a complex feedback system of dynamic and reciprocal signaling. We argue that it is through this framework that we can start to address one of the central challenges in breast cancer research to understand the molecular mechanisms driving progression from benign to invasive.As molecular profiling of Ductal Carcinoma In Situ (DCIS) and Invasive Ductal Carcinoma (IDC) have shown no robust genetic differences between the cancers, it has been argued that invasiveness is not a property acquired by the tumor, but rather a functional consequence of the loss of tumor-suppressive properties of the underlying supporting cells, specifically the myoepithelial cells that lie basally to the tumor cell-of-origin. Myoepithelial cells have been shown to be key regulators of normal tissue architecture, and dysregulation in their function can lead to loss of apico-basal polarity of luminal cells and changes in the deposition of basement membrane components (Gudjonsson, T., et al. 2002). Furthermore, in a xenograft model of DCIS, normal myoepithelial cells have been shown to have tumor-­suppressive properties, able to delay both tumor growth and acquisition of an invasive phenotype (Hu, M., et al. 2008). On the other hand, a transcriptomic profiling study comparing different cell populations of the breast between normal, DCIS and IDC shows that the earliest genetic changes occurring between normal and DCIS tissue arise in the myoepithelial lineage – largely through the aberrant upregulation of genes for secreted factors including matrix-remodeling enzymes, tumor-promoting cytokines and signaling factors (Allinen, M., et al. 2004). Thus, myoepithelial cells undergo a switch from tumor-suppressive to tumor-promoting behavior from normal to the earliest stage of cancer. When do these changes in the myoepithelial lineage begin to occur and what are the driving forces behind these changes? As changes in normal mammary gland architecture and its cellular and molecular composition are already apparent in aged breast tissue, including loss of lineage specificity in the epithelial lineages (Garbe, J.C., et al. 2012) (Benz, C.C. 2008), we propose that the underlying changes in the normal function of myoepithelial cells start to occur as early as aging, and play a key role in the processes underlying age-related susceptibility to breast cancer. Furthermore, we hypothesize that changes in the microenvironment that accompany aging and tumor progression drive these changes in the myoepithelial lineage through the process of “dynamic reciprocity,” where dynamic and reciprocal signaling between the altered myoepithelial microenvironment and the myoepithelial cells lead to further disruption of proper function of the lineage. To address these questions, we have (1) undertaken comprehensive transcriptomic profiling studies of myoepithelial cells in the context of normal primary breast tissue, and of an established human mammary epithelial cell (HMEC) culture system that is able to maintain the transcriptional, biochemical, and phenotypic features of lineage and chronological age (Garbe, J.C., et al. 2012); and (2) designed relatively simple yet biologically-relevant culture systems that allow for functional studies of myoepithelial cells in “young” vs. “aged” systems, and in “normal-like” vs. “tumor-like” microenvironments to illustrate how aberrations in normal myoepithelial function can lead to the changes we observe in aging and cancer.Our work to develop analytical methodologies that aim to move gene-level and cell-level analysis into a systems-level approach have generated a complex profile of the myoepithelial lineage: (1) Our transcriptomic profiling of primary myoepithelial cells, along with the other major cell populations of the breast, has elucidated major characteristics of myoepithelial cells including upregulated lineage-specific genes and their associated pathways, as well as potential cell-cell interactions and co-regulatory networks between myoepithelial cells and other cell populations in the breast, and their contribution to the maintenance of the tissue microenvironment; (2) Our transcriptomic profiling of myoepithelial cells isolated from HMEC derived from a cohort of young pre-menopausal and older post-menopausal women have identified key pathways, regulatory networks and transcription factors dysregulated in the myoepithelial lineage during aging. While our work to design biologically-relevant cell culture systems has revealed key insights into the phenotypic changes observed in aging and cancer: (1) our “young” and “aged” co-culture system illustrates the ability of myoepithelial cells to confer an age-specific phenotype to luminal cells; and (2) our “normal-like” and “tumor-like” culture system demonstrates the ability of extracellular matrix (ECM) proteins to differentially regulate the myoepithelial secretome, with “tumor-like” ECM inducing myoepithelial secretion of angiogenic, cancer-associated factors, and matrix remodeling enzymes that mimic their aberrant secretory behavior in DCIS.One the standing challenges of breast cancer research is to understand the underlying molecular mechanisms driving progression of breast cancer from benign to invasive. While progress has been made to identify tumor suppressor genes and oncogenes, as well as genetic alterations that associate with certain cancer subtypes, the correlation of these genotypic-phenotypic events is not fully understood. This poses a problem not only in understanding the biology of cancer, but equally significant, in developing reliable prognostic markers of progression and recurrence, as well as designing treatment options for women. Our current understanding of progression implicates myoepithelial cells as a major driver regulating transition to invasiveness. Thus, our understanding of how normal myoepithelial cells function, and how they become dysregulated in biologically-relevant model systems of aged and tumor-like microenvironments can provide insight into the molecular mechanisms which underlie the loss of normal function of the myoepithelial lineage that drive cancer progression

Changes in Immune Cell Types with Age in Breast are Consistent with a Decline in Immune Surveillance and Increased Immunosuppression

A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based image analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary breast tissue preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm2) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2 macrophage density was higher than M1 macrophage density and this difference was due to higher density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age

Discriminating External and Internal Causes for Heading Changes in Freely Flying <em>Drosophila</em>

<div><p>As animals move through the world in search of resources, they change course in reaction to both external sensory cues and internally-generated programs. Elucidating the functional logic of complex search algorithms is challenging because the observable actions of the animal cannot be unambiguously assigned to externally- or internally-triggered events. We present a technique that addresses this challenge by assessing quantitatively the contribution of external stimuli and internal processes. We apply this technique to the analysis of rapid turns (“saccades”) of freely flying <i>Drosophila melanogaster</i>. We show that a single scalar feature computed from the visual stimulus experienced by the animal is sufficient to explain a majority (93%) of the turning decisions. We automatically estimate this scalar value from the observable trajectory, without any assumption regarding the sensory processing. A posteriori, we show that the estimated feature field is consistent with previous results measured in other experimental conditions. The remaining turning decisions, not explained by this feature of the visual input, may be attributed to a combination of deterministic processes based on unobservable internal states and purely stochastic behavior. We cannot distinguish these contributions using external observations alone, but we are able to provide a quantitative bound of their relative importance with respect to stimulus-triggered decisions. Our results suggest that comparatively few saccades in free-flying conditions are a result of an intrinsic spontaneous process, contrary to previous suggestions. We discuss how this technique could be generalized for use in other systems and employed as a tool for classifying effects into sensory, decision, and motor categories when used to analyze data from genetic behavioral screens.</p> </div